Mammalian erythropoiesis is normally regulated in vivo by Epo (erythropoietin), a glycoprotein hormone responsible for the differentiation of red blood cells and for the viability and proliferation of their progenitors. Years after the introduction of culture media for the growth of the erythroid progenitors CFU-E (colony forming unit-erythroid) and BFU-E (burst forming unit-erythroid) it was found that fetal bovine serum (FBS) contained significant quantities of an Epo-like activity..sup.1 Originally, this was believed to be due to the presence of Epo in FBS, but radioimmunoassays of Epo in FBS found only minute amounts of the hormone..sup.2,3 Subsequently, it was shown that the Epo-like activity of FBS was abrogated by an antiserum directed against insulin-like growth factor I(IGF-I), but not by anti-Epo antiserum..sup.3 The same investigators also found that purified human IGF-I could stimulate colony-formation by CFU-E from murine fetal liver and adult bone marrow cells in a "serum free" "SF" medium..sup.4 Further studies showed that IGF-I enhanced both CFU-E- and BFU-E-derived colony formation from human bone marrow and peripheral blood in "SF" medium as well.sup.5.
To determine the cellular and molecular mechanisms that regulate erythropoiesis, there is needed a truly SF culture system for circulating erythroid progenitor cells which would be defined with respect to all activities that are stimulatory for erythropoiesis. Simple depletion of serum from a culture medium does not necessarily remove from it all undefined serum factors. Major vehicles for the uncontrolled presence of such factors in culture media, in the absence of added serum or plasma, are the albumin preparations commonly employed, most typically Cohn's Fraction V. These have been shown to provide a mitogenic factor(s).sup.6,7 referred to as BPA ("Burst Promoting Activity".sup.8) which has so far remained molecularly unidentified. In fact, most so-called "SF" media in the current literature use bovine serum albumin (BSA) preparations that are known to be contaminated with such activities. Effectively, serum residues provide a "dirty background" which precludes accurate and reliable study of the precise effects of added growth factors. Accordingly, in this specification, the term "serum-free" or "SF" is used in quotation marks in instances where reference is made to a medium reported originally to be serum-free but subsequently discovered to be still serum-contaminated.
In our laboratory, we have previously shown that treatment of BSA with activated charcoal removes its BPA-like activity..sup.6 Using a fatty-acid-free and globulin-free BSA (FAF, GF BSA) preparation in a SF medium for the growth of BFU-E from bone marrow, Ogawa and co-workers.sup.9 demonstrated that under these stringent conditions, a defined exogenous source of BPA-like activity, interleukin-3 (IL-3) or GM-CSF, had to be added in order to obtain growth of day-14 erythroid bursts. With this SF medium, however, we were unable to grow erythroid bursts or-colonies from circulating progenitor cells.
The study of erythropoiesis, including the study of the growth and proliferation of leukemia cells, so important to the development of understanding and eventual control of this disease, requires the provision of a cell growth medium which ensures optimum growth of the cells under study. It also requires the provision of a cell growth medium free from unknown ingredients which might affect the cell growth characteristics. Only with such a growth medium can the researcher satisfactorily study the effects of other growth-promoting or -inhibiting factors on cell proliferation and differentiation, as well as study the precise effects of toxic molecules.
It is an object of the present invention to provide a novel cell culture medium for hemopoietic and leukemia cells.